Melanoma is a form of cancer that develops from pigment-producing cells called melanocytes and is responsible for 80 % of skin cancer-related deaths. Given its ability to spread and metastasize at high rates to multiple sites, it is identified as one of the most aggressive human tumors [1]. More than 50 % of cutaneous melanoma cases undergo BRAF mutations rendering it to be a key driver in melanoma progression. Amongst these, BRAF V600E mutation occurs most frequently conferring subsequent activation of the BRAF kinase activity. This hotspot mutation led to the revolution of molecular targeted therapies for BRAF mutant melanoma. The FDA approved vemurafenib (Vem) in 2011 as the first potent BRAF inhibitor (BRAFi) for patients with advanced melanoma. Treatment with Vem initially resulted in excellent response and prolonged survival as compared to chemotherapeutic agents. However, within an average period of 7 months, patients eventually relapse due to rapid onset of resistance against Vem thereby limiting its long-term clinical success. As a result, combination therapy of BRAFi and MEKi like trametinib was approved in 2017 by the FDA to forestall the development of BRAFi resistance in melanoma patients. However, several setbacks were encountered including development of cross-resistance between BRAFi and MEKi and off-target toxicities [2].

Several mechanisms accountable for development of resistance against Vem have been discovered by employing in vitro and in vivo models, as well as in pre- and post-treatment tumor samples of melanoma patients. Preceding studies have revealed that reactivation of the Mitogen-activated protein kinase (MAPK) pathway most commonly occurs in BRAFi-resistant tumors, while the second majorly activated pathway is phosphoinositide-3-kinase–protein kinase B/Akt (PI3K-PKB/Akt). MAPK reactivation induces alterations in NRAS, BRAF, and MEK, and increased PI3K/AKT signaling is often associated with the loss of PTEN tumor suppressor gene or upregulation of receptor tyrosine kinases (RTKs) [3]. Researchers have also proven the role of Rho-mediated signaling in promoting BRAF inhibitor resistance in melanoma cells [4,5]. Hyperactivation of these key signaling pathways converge towards activation of oncogenic c-Myc to develop intrinsic as well as acquired resistance against BRAFi like Vem [6]. Intrinsic resistance is the innate resistance against Vem that exists before its exposure to cause reduced antitumor efficacy, while acquired resistance can be recognized by gradual decrease in anticancer efficacy of Vem following several treatment cycles [7]. Development of resistance against BRAFi not only results in genomic or epigenetic abnormalities, but also poses a major effect on the tumor microenvironment in melanoma. Treatment with Vem causes direct activation of cancer-associated fibroblasts (CAFs), which in turn leads to reactivation of both the MAPK/ERK and PI3K/AKT signaling pathways for prolonged survival. Moreover, melanoma cells in the neighborhood of CAFs present an aggressive phenotype due to increased abundance of the extracellular matrix (ECM) proteins including collagen, integrins, and enhanced TGF-β levels. Such tumor microenvironment properties of melanoma resistant to targeted therapies favor tumor cell proliferation, adhesion, and invasiveness resulting in even highly aggressive and invasive cancer with elevated metastatic potential [8].

Since drug resistance remains a major challenge in melanoma therapy, researchers have been have been investigating more effective anticancer therapeutics aimed to fight and overcome the resistance. To serve that purpose, all potential antitumor candidates must be evaluated in in vitro cell culture models initially before being tested at preclinical stages in vivo and further in human clinical trials. Establishment of drug-resistant tumor cell lines has been previously employed by various researchers to identify possible underlying mechanisms that promote cancer progression as well as to evaluate anticancer efficacy of novel single or combination therapeutic cargoes including drugs, nucleic acids or monoclonal antibodies. Drug-resistant cell lines established from parental cell lines stand as the most useful models in cancer research and are developed either by stepwise dose-escalation continuous exposure or by high-concentration pulsatile exposure approach [9]. Apart from two-dimensional (2D) cell culture models, multicellular tumor spheroids (MCTS) have evolved as in vitro three‐dimensional (3D) cell culture models in cancer research since they provide structurally comparable conditions to that present in in vivo tumor environment. 3D MCTS closely mimic the critical features of human solid tumors in terms of tumor heterogeneity, structural organization, hypoxia, cellular layered assembling, and nutrient gradients. These properties impart an anticancer therapeutics resistance profile to 3D MCTS, which is identical to that exhibited by human solid tumors. Also, deposition of ECM components, cell to cell and cell to ECM interactions can only be observed in 3D spheroids model rendering them to be a more pertinent in vitro model when it comes to developing clinically relevant anticancer therapeutics, especially for drug-resistant tumors [10,11]. In this context, we generated and comprehensively characterized two BRAF V600E mutant melanoma cell lines, A375 and SK-MEL-28, with acquired resistance to Vem. Additionally, we performed detailed characterization of an intrinsically resistant melanoma cell line, RPMI-7951. The in vitro characterization was carried out in both 2D and 3D models of Vem-resistant melanoma. In characterizing 2D models, we examined changes in the cellular morphology, growth rate, migration, colony forming ability, and apoptosis in parental and resistant cell lines. While 3D characterization involved development of spheroids, assessing the growth rate and invasion in both parental and resistant cell lines. We also evaluated the anticancer activity portrayed by a chemotherapeutic drug, docetaxel, which acts as a microtubular depolymerization inhibitor, in comparison to Vem as a BRAFi in developed resistant 3D cell culture model. Importantly, we verified the resistance of developed A375V cell line against Vem in vivo to find negligible tumor growth inhibition following Vem treatment in tumor xenograft model. Therefore, for the very first time, we have established and systematically characterized melanoma cells with both acquired and intrinsic resistance to BRAFi to identify the growth rate, invasiveness, and metastatic potential of resulting 2D and 3D resistant melanoma models. Resistance development in established acquired BRAFi-resistant cell line was also validated in in vivo model. We believe that construction of novel drug-resistant melanoma models and identification of their behavior in vitro as well as in vivo would aid in designing and screening clinically translational anticancer therapeutics to combat Vem resistance in melanoma.